Tissue culture techniques are being used in developing tissue and organ equivalents. These techniques involve collagen matrix structures which are capable of being remodeled into functional tissue and organs by the right combination of living cells, nutrients, and culturing conditions.
Currently the use of cultured tissue equivalents is restricted because such equivalents have a limited shelf-life. Thus, at present it is impossible to maintain a proper inventory for any length of time. Further, because of the limited shelf-life of the equivalents, there is a high degree of waste. Accordingly, the development of a successful cryopreservation method which would extend the length of time that the cultured tissues could be stored so that a proper inventory could be maintained, and which would substantially reduce waste, is desirable. Such a cryopreservation method would enable the commercial use of such equivalents since an inventory of larger batch sizes could be maintained and quality control inspected. Currently, USP standards mandate sterility testing which requires 14 days. An additional advantage to developing a successful cryopreservation method is that it would enable one to arrest the growth of the cultured tissue equivalent at specific stages of development for further testing and analysis.
Currently, the storage time of biological materials is extended by freezing. The solidification of a liquid by freezing can take place as crystallization, involving an orderly arrangement of molecules. Alternatively, freezing can take place as amorphization or vitrification, a solidification without such an orderly arrangement.
Freezing living cells by crystallization is problematic in that intracellular ice crystals are formed. These ice crystals are detrimental to cell viability upon thawing. The cells could survive solidification and thawing, however, if they are cryopreserved by vitrification. Vitrification involves cooling and warming cells at controlled rates while the cells are immersed in cryoprotectants.
Fahy et al., "Vitrification as an approach to cryopreservation," Cryobiology 21:407-426 (1984), developed a cryoprotectant solution containing dimethyl sulfoxide (DMSO) and polymers which vitrified at 1 atmosphere. This cryopreservation medium was modified for the vitrification of human monocytes. Takahashi et al., "Vitrification of Human Monocytes," Cryobiology 23:103-115 (1986).
Vitrification of cells and tissues using cryoprotectants is problematic in that cell viability is low upon thawing because the cells cannot tolerate the highly concentrated cryoprotectant solutions used, the high rates of cooling and warming, thermal shock, and ice formation during warming or devitrification. Using prior art methods, it is not possible to cryopreserve a cultured tissue equivalent, in part because it is relatively thick and very heterogeneous. The present inventors have discovered that the same cryopreservation approach cannot be applied to all tissues. Thus, at present there is a strong need for an effective and commercially practical method of cyropreservation for cultured tissue equivalents.